Epitope tagging of chromosomal genes in <i>Salmonella</i>

We have developed a simple and efficient procedure for adding an epitope-encoding tail to one or more genes of interest in the bacterial chromosome. The procedure is a modification of the gene replacement method of Datsenko and Wanner [Datsenko, K. A. &amp; Wanner, B. L. (2000) Proc. Natl. Acad. Sci. USA 97, 6640–6645]. A DNA module that begins with the epitope-encoding sequence and includes a selectable marker is amplified by PCR with primers that carry extensions (as short as 36 nt) homologous to the last portion of the targeted gene and to a region downstream from it. Transformation of a strain expressing bacteriophage ʎ red functions yields recombinants carrying the targeted gene fused to the epitope-encoding sequence. The resulting C-terminal-tagged protein can be identified by standard immuno-detection techniques. In an initial application of the method, we have added the sequences encoding the FLAG and 3xFLAG and influenza virus hemagglutinin epitopes to various genes of Salmonella enterica serovar Typhimurium, including putative and established pathogenic determinants present in prophage genomes. Epitope fusion proteins were detected in bacteria growing in vitro, tissue culture cells, and infected mouse tissues. This work identified a prophage locus specifically expressed in bacteria growing intracellularly. The procedure described here should be applicable to a wide variety of Gram-negative bacteria and is particularly suited for the study of intracellular pathogens

Insertion hot spot for horizontally acquired DNA within a bidirectional small-RNA locus in Salmonella enterica

In Escherichia coli and Salmonella enterica, RyeA and RyeB RNAs are encoded on opposite DNA strands at the same locus. We present evidence indicating that the last 23 bp of the ryeB gene, corresponding to an internal portion of the ryeA gene, served repeatedly as the integration site for exogenous DNA during Salmonella evolution and still act as an attachment site for present-day bacteriophages. Interestingly, ryeA sequence and expression are modified upon lysogenization.Ministerio de Educación y Ciencia BIO2004-3455-CO2-0

The tripartite capsid gene of Salmonella phage Gifsy-2 yields a capsid assembly pathway engaging features from HK97 and λ

AbstractPhage Gifsy-2, a lambdoid phage infecting Salmonella, has an unusually large composite gene coding for its major capsid protein (mcp) at the C-terminal end, a ClpP-like protease at the N-terminus, and a ∼200 residue central domain of unknown function but which may have a scaffolding role. This combination of functions on a single coding region is more extensive than those observed in other phages such as HK97 (scaffold–capsid fusion) and λ (protease–scaffold fusion). To study the structural phenotype of the unique Gifsy-2 capsid gene, we have purified Gifsy-2 particles and visualized capsids and procapsids by cryoelectron microscopy, determining structures to resolutions up to 12Å. The capsids have lambdoid T=7 geometry and are well modeled with the atomic structures of HK97 mcp and phage λ gpD decoration protein. Thus, the unique Gifsy-2 capsid protein gene yields a capsid maturation pathway engaging features from both phages HK97 and λ

Prophage Gifsy-1 Induction in Salmonella enterica Serovar Typhimurium Reduces Persister Cell Formation after Ciprofloxacin Exposure

Persister cells are drug-tolerant bacteria capable of surviving antibiotic treatment despite the absence of heritable resistance mechanisms. It is generally thought that persister cells survive antibiotic exposure through the implementation of stress responses and/or energy-sparing strategies. Exposure to DNA gyrase-targeting antibiotics could be particularly detrimental for bacteria that carry prophages integrated in their genomes. Gyrase inhibitors are known to induce prophages to switch from their dormant lysogenic state into the lytic cycle, causing the lysis of their bacterial host. However, the influence of resident prophages on the formation of persister cells has only been recently appreciated. Here, we evaluated the effect of endogenous prophage carriage on the generation of bacterial persistence during Salmonella enterica serovar Typhimurium exposure to both gyrase-targeting antibiotics and other classes of bactericidal antibiotics. Results from the analysis of strain variants harboring different prophage combinations revealed that prophages play a major role in limiting the formation of persister cells during exposure to DNA-damaging antibiotics. In particular, we present evidence that prophage Gifsy-1 (and its encoded lysis proteins) are major factors limiting persister cell formation upon ciprofloxacin exposure. Resident prophages also appear to have a significant impact on the initial drug susceptibility, resulting in an alteration of the characteristic biphasic killing curve of persister cells into a triphasic curve. In contrast, a prophage-free derivative of S. Typhimurium showed no difference in the killing kinetics for β-lactam or aminoglycoside antibiotics. Our study demonstrates that induction of prophages increased the susceptibility toward DNA gyrase inhibitors in S. Typhimurium, suggesting that prophages have the potential for enhancing antibiotic efficacy

Induction and migration of cryptic/defective Salmonella enterica prophages as a consequence of infection with lytic phages is an additional factor in stability of a coevolutionary vector

The influence of infection of natural isolates of Salmonella enterica with lytic (nonlysogenic) phages on the expression of resident cryptic or defective prophages in host bacteria was studied. The induction of defective/cryptic phages after infection with nonlysogenic phages and packaging of bacterial chromosomal fragments in capsids of defective phages is demonstrated. This may lead to migration and wide distribution of both the genomes of defective phages per se and various fragments of the bacterial chromosome (including pathogenic islands) in new bacterial strains with concomitant change of their properties, the acquired new features of pathogenicity among them.This work was supported by EC PhageVet-P (contract no. FOOD-CT-2005-007224) and by the Russian Foundation for Basic Research (grant no. 08-04-00162-a). We gratefully acknowledge the support of organizations presenting the grants.info:eu-repo/semantics/publishedVersio

Salmonella Enteritidis bacteriophage candidates for phage therapy of poultry

Aims: Salmonella is a worldwide foodborne pathogen causing acute enteric infections in humans. In the recent years, the use of bacteriophages has been suggested as a possible tool to combat this zoonotic pathogen in poultry farms. This work aims to isolate and perform comparative studies of a group of phages active against a collection of specific Salmonella Enteritidis strains from Portugal and England. Also, suitable phage candidates for therapy of poultry will be selected. Methods and Results: The Salm. Enteritidis strains studied were shown to have a significantly high occurrence of defective (cryptic) prophages; however, no live phages were found in the strains. Bacteriophages isolated from different environments lysed all except one of the tested Salm. Enteritidis strains. The bacteriophages studied were divided into different groups according to their genetic homology, RFLP profiles and phenotypic features, and most of them showed no DNA homology with the bacterial hosts. The bacteriophage lytic efficacy proved to be highly dependent on the propagation host strain. Conclusions: Despite the evidences shown in this work that the Salm. Enteritidis strains used did not produce viable phages, we have confirmed that some phages, when grown on particular hosts, behaved as complexes of phages. This is most likely because of the presence of inactive phage-related genomes (or their parts) in the bacterial strains which are capable of being reactivated or which can recombine with lytic phages. Furthermore, changes of the bacterial hosts used for maintenance of phages must be avoided as these can drastically modify the parameters of the phage preparations, including host range and lytic activity. Significance and Impact of the Study: This work shows that the optimal host and growth conditions must be carefully studied and selected for the production of each bacteriophage candidate for animal therapy.The authors kindly acknowledge the Instituto Nacional Ricardo Jorge (INRJ) (Portugal) and also Tom Humphrey from the Division of Farm Animal Science, School of Clinical Veterinary Science of the University of Bristol (UK) for providing the Salm. Enteritidis isolates used in this work

Bacteriophage Crosstalk: Coordination of Prophage Induction by Trans-Acting Antirepressors

Many species of bacteria harbor multiple prophages in their genomes. Prophages often carry genes that confer a selective advantage to the bacterium, typically during host colonization. Prophages can convert to infectious viruses through a process known as induction, which is relevant to the spread of bacterial virulence genes. The paradigm of prophage induction, as set by the phage Lambda model, sees the process initiated by the RecA-stimulated self-proteolysis of the phage repressor. Here we show that a large family of lambdoid prophages found in Salmonella genomes employs an alternative induction strategy. The repressors of these phages are not cleaved upon induction; rather, they are inactivated by the binding of small antirepressor proteins. Formation of the complex causes the repressor to dissociate from DNA. The antirepressor genes lie outside the immunity region and are under direct control of the LexA repressor, thus plugging prophage induction directly into the SOS response. GfoA and GfhA, the antirepressors of Salmonella prophages Gifsy-1 and Gifsy-3, each target both of these phages' repressors, GfoR and GfhR, even though the latter proteins recognize different operator sites and the two phages are heteroimmune. In contrast, the Gifsy-2 phage repressor, GtgR, is insensitive to GfoA and GfhA, but is inactivated by an antirepressor from the unrelated Fels-1 prophage (FsoA). This response is all the more surprising as FsoA is under the control of the Fels-1 repressor, not LexA, and plays no apparent role in Fels-1 induction, which occurs via a Lambda CI-like repressor cleavage mechanism. The ability of antirepressors to recognize non-cognate repressors allows coordination of induction of multiple prophages in polylysogenic strains. Identification of non-cleavable gfoR/gtgR homologues in a large variety of bacterial genomes (including most Escherichia coli genomes in the DNA database) suggests that antirepression-mediated induction is far more common than previously recognized

Regulation of the small regulatory RNA MicA by ribonuclease III: a target-dependent pathway

MicA is a trans-encoded small non-coding RNA, which downregulates porin-expression in stationary-phase. In this work, we focus on the role of endoribonucleases III and E on Salmonella typhimurium sRNA MicA regulation. RNase III is shown to regulate MicA in a target-coupled way, while RNase E is responsible for the control of free MicA levels in the cell. We purified both Salmonella enzymes and demonstrated that in vitro RNase III is only active over MicA when in complex with its targets (whether ompA or lamB mRNAs). In vivo, MicA is demonstrated to be cleaved by RNase III in a coupled way with ompA mRNA. On the other hand, RNase E is able to cleave unpaired MicA and does not show a marked dependence on its 5′ phosphorylation state. The main conclusion of this work is the existence of two independent pathways for MicA turnover. Each pathway involves a distinct endoribonuclease, having a different role in the context of the fine-tuned regulation of porin levels. Cleavage of MicA by RNase III in a target-dependent fashion, with the concomitant decay of the mRNA target, strongly resembles the eukaryotic RNAi system, where RNase III-like enzymes play a pivotal role

Invasive meningococcal disease in three siblings with hereditary deficiency of the 8th component of complement: Evidence for the importance of an early diagnosis

Deficiency of the eighth component of complement (C8) is a very rare primary immunodeficiency, associated with invasive, recurrent infections mainly caused by Neisseria species. We report functional and immunochemical C8 deficiency diagnosed in three Albanian siblings who presented with severe meningococcal infections at the age of 15 years, 4 years and 17 months, respectively. The youngest suffered serious complications (necrosis of fingers and toes requiring amputation). METHODS: Functional activity of the classical, alternative and mannose-binding lectin complement pathways was measured in serum from the 3 siblings and their parents (37-year-old woman and 42-year-old man). Forty healthy subjects (20 males and 20 females aged 4-38 years) served as normal controls. Serum complement factors were measured by haemolytic assays and immunoblotting. Sequence DNA analysis of the C8B gene was performed. RESULTS: Analyses of the three complement pathways revealed no haemolytic activity and also absence of C8beta in serum samples from all three siblings. The genetic analysis showed that the three siblings were homozygous for the p.Arg428* mutation in the C8B gene on chromosome 1p32 (MIM 120960). The parents were heterozygous for the mutation and presented normal complement activities. A 2-year follow-up revealed no further infective episodes in the siblings after antibiotic prophylaxis and meningococcal vaccination. CONCLUSIONS: Complement deficiencies are rare and their occurrence is often underestimated. In presence of invasive meningococcal infection, we highlight the importance of complement screening in patients and their relatives in order to discover any genetic defects which would render necessary prophylaxis to prevent recurrent infections and severe complications

A new Vibrio cholerae sRNA modulates colonization and affects release of outer membrane vesicles

We discovered a new small non-coding RNA (sRNA) gene, vrrA of Vibrio cholerae O1 strain A1552. A vrrA mutant overproduces OmpA porin, and we demonstrate that the 140 nt VrrA RNA represses ompA translation by base-pairing with the 5′ region of the mRNA. The RNA chaperone Hfq is not stringently required for VrrA action, but expression of the vrrA gene requires the membrane stress sigma factor, σE, suggesting that VrrA acts on ompA in response to periplasmic protein folding stress. We also observed that OmpA levels inversely correlated with the number of outer membrane vesicles (OMVs), and that VrrA increased OMV production comparable to loss of OmpA. VrrA is the first sRNA known to control OMV formation. Moreover, a vrrA mutant showed a fivefold increased ability to colonize the intestines of infant mice as compared with the wild type. There was increased expression of the main colonization factor of V. cholerae, the toxin co-regulated pili, in the vrrA mutant as monitored by immunoblot detection of the TcpA protein. VrrA overproduction caused a distinct reduction in the TcpA protein level. Our findings suggest that VrrA contributes to bacterial fitness in certain stressful environments, and modulates infection of the host intestinal tract